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. 2008 Apr;19(4):1783–1797. doi: 10.1091/mbc.E07-07-0646

Figure 5.

Figure 5.

Defective inward translocation of NBD-labeled phospholipids in the fpk1Δ fpk2Δ mutant. (A) Uptake of NBD-labeled phospholipids. Cells were preincubated in SC medium for 3 h at 30°C and labeled with NBD-PE, -PC, or -PS for 30 min at 30°C, and internalized NBD-phospholipids were quantitated by flow cytometry as described in Materials and Methods. Strains used were as follows: top panel, KKT70 (WT), KKT102 (lem3Δ), KKT72 (fpk1Δ), KKT266 (fpk2Δ), and KKT268 (fpk1Δ fpk2Δ); bottom panel, the wild-type (WT) strain transformed with YCplac111 (vector) and the fpk1Δ fpk2Δ mutant transformed with YCplac111 (vector), YCplac111-FPK1 (pFPK1), or YCplac111-FPK1(K525R) [pFPK1(K525R)]. Data are presented as average percentage of accumulation relative to wild-type cells ± SD of three independent experiments. (B) Efflux of NBD-PE and NBD-PS was not affected in the fpk1Δ fpk2Δ mutant. Phospholipid efflux in wild-type (KKT70, •), lem3Δ (KKT102, ○), and fpk1Δ fpk2Δ (KKT268, □) cells was measured at the indicated times as described in Materials and Methods. Relative values for the initial fluorescence in the lem3Δ mutant were 91.6 ± 1.3 and 90.5 ± 16.5% of the wild type for NBD-PE and -PS, respectively, and those in the fpk1Δ fpk2Δ mutant were 96.3 ± 12.4 and 91.2 ± 3.0%, respectively. Data are presented as means ± SD of at least three independent experiments. (C) Simultaneous overexpression of DNF1 and LEM3 suppressed the duramycin-sensitive growth of the fpk1Δ fpk2Δ mutant. fpk1Δ fpk2Δ cells (KKT268) harboring YEplac181 and YEplac195 (vector), YEplac181 and YEplac195-FPK1 (pFPK1), or YEplac181-LEM3 and YEplac195-DNF1 (pDNF1/LEM3) were tested for duramycin sensitivity as described in the legend of Figure 4A. (D) Simultaneous overexpression of DNF1 and LEM3 restored the uptake of NBD-labeled phospholipids in the fpk1Δ fpk2Δ mutant. Wild-type (WT) and fpk1Δ fpk2Δ cells harboring YEplac181 and YEplac195 (vector) or YEplac181-LEM3 and YEplac195-DNF1 (pDNF1/LEM3) were assayed for NBD-phospholipid internalization. Results are presented as described in A.